CHROMATOGRAPHY Flashcards
is a technique in which the components of a mixture are separated based on differences in the rates at which they are carried through a fixed or stationary phase by a gaseous or liquid mobile phase
Chromatography
It is a phase that is fixed in place either in a column or on a planar surface
Stationary Phase
It is the phase that moves over or through the stationary phase carrying with it the analyte mixture. - It can be gas, liquid or a supercritical fluid
Mobile Phase
Stationary phase is held in a narrow tube
The mobile phase is forced through the tube under pressure or by gravity
Column Chromatography
Used for the separation of steroids, barbiturates, blood alcohol and lipids
Gas Chromatography
Two Types of Gas Chromatography
Gas Solid Chromatography
Gas Liquid Chromatography
Measure differences in absorption at the solid phase surface
the mobile phase is a gas, and the stationary phase is a solid that retains the analytes by physical adsorption
Gas Solid Chromatography -
Separation occurs by differences in solute partitioning between the gaseous mobile phase and the liquid stationary phase
the mobile phase is a gas, and the stationary phase is a liquid that is retained on the surface of an inert solid by adsorption or chemical bonding
Gas Liquid Chromatography
Mobile phase in gas chromatography
Chemically inert
Carrier Gas System -
is the most common mobile phase (argon, nitrogen, and hydrogen are also used)
Helium
is widely used measure the desirable flow through the column.
Classical Soap Bubble -
For high column efficiency, a suitably sized sample should be introduced as a “plug” of vapor.
Sample Injection System
is achieved by increasing the column temperature continuously or in steps during elution
TEMPERATURE PROGRAMMING
Most widely used and generally applicable detector for gas chromatography
Effluent from the column is directed into a small air/hydrogen flame
Flame Ionization Detector (FID)
one of the earliest detectors for gas chromatography
Consist of an electrically heated source whose temperature at constant electric power depends on the thermal conductivity of the surrounding gas
Thermal Conductivity Detector (TCD)
Most widely used detectors for environmental samples
Selectively responds to halogen – containing organic compounds (pesticides and polychlorinated biphenyls)
Electron Capture Detector (ECD)
One of the most powerful detectors for gas chromatography
Based on the fragmentation and ionization of molecules using a suitable source of energy
Mass Spectrometry Detectors
Gold standard for drug testing
Uses an electron beam to split the drug emerging from the column into its component ions
Gas Chromatography – Mass Spectrometry (GC/MS)
Can detect 20 inborn errors of metabolism from a single spot
Tandem Mass Spectroscopy (MS/MS)
Also called open tubular columns
Capillary Column
Capillary tubes coated with a thin layer of the liquid stationary phase
Columns are made of stainless steal, aluminum. Copper, or plastic
Wall – Coated Open Tubular (WCOT)
The inner surface of the capillary is lined with a thin film of a solid support material on which the liquid stationary phase is adsorbed
Support – Coated Open Tubular (SCOT)
Currently the most widely used GC columns.
Fused – Silica Open Tubular Capillaries
Modern packed columns are fabricated from glass or metal tubing.
Typically 2 to 3 m long and have inside diameters of 2 to 4 mm.
Packed Columns
Widely used to establish the purity of organic compounds
Qualitative Analysis
Based on comparison of either the height or the area of an analyte peak with that of one or more standards.
Quantitative Analysis
Based on the distribution of solutes between a liquid mobile phase and a stationary phase.
LIQUID CHROMATOGRAPHY
is the most widely used liquid chromatography.
HPLC
Used for the fractionation of drugs, hormones, lipids, carbohydrates and proteins.
HPLC (high Performance Liquid Chromatography)
Separates molecules based on differences in their size and shape
Gel/ Gel Permeation/ Gel Filtration/ Size Exclusion/ Molecular Sieve Chromatography
for separation of enzymes, antibodies and proteins - Examples: DEXTRAN and AGAROSE
Hydrophilic Gel
for separation of triglyceride and fatty acid - Example: sephadex
Hydrophobic Gel
Separation of nucleic acid and proteins depends primarily on the sign and ionic charge density.
For separation of amino acids, proteins and nucleic acids.
Ion Exchange Chromatography
Separation is based on relative solubility in an organic (nonpolar) solvent and an aqueous (polar solvent)
For separation of therapeutic drugs and their metabolites
Partition Chromatography (Liquid – Liquid Chromatography)
Used to separate and prepare larger quantities of proteins and antibodies for study
For separation of lipoproteins, Carbohydrates and glycated hemoglobin
Affinity Chromatography
Separation is based on the differences between the adsorption and desorption of solutes at the surface of a solid particle
Adsorption Chromatography
based on adsorption of gaseous substances on solid surfaces
Gas – Solid Chromatography